DNA interaction, free radical scavenging and in-vitro antibacterial activity of drug-based copper(II) complexes

Article abstract of DOI:10.1002/aoc.1817

The Cu(II) complexes of type [Cu(cpf)(Aⁿ)Cl] (Aⁿ = terpyridines, cpf = ciprofloxacin) were synthesized and characterized using IR, mass and reflectance spectra. The free ligands and their complexes were evaluated for their in-vitro antimicrobial activity against a panel of Gram-positive and Gram-negative bacteria. The complexes exhibit better or equal inhibition in comparison to free fluoroquinolones. Binding interactions of the complexes with calf thymus (CT DNA) were investigated by absorption titration, viscosity studies and DNA melting temperature experiment. The cleavage reaction on pUC19 DNA was monitored by agarose gel electrophoresis. The lower concentration of the complexes was catalysed the dismutation of superoxide radical at biological pH, which indicates that the complexes can act as a possible model for superoxide dismutase. Copyright

Full text of DOI:10.1002/aoc.1817

Full Paper
Received: 27 January 2011
Revised: 13 May 2011
Accepted: 22 May 2011
Published online in Wiley Online Library: 10 August 2011
(wileyonlinelibrary.com) DOI 10.1002/aoc.1817
DNA interaction, free radical scavenging
and in-vitro antibacterial activity of drug-based
copper(II) complexes
∗
Mohan N. Patel , Promise A. Dosi and Bhupesh S. Bhatt
The Cu(II) complexes of type [Cu(cpf)(A )Cl] (An = terpyridines, cpf = ciprofloxacin) were synthesized and characterized using
IR, mass and reflectance spectra. The free ligands and their complexes were evaluated for their in-vitro antimicrobial activity
against a panel of Gram-positive and Gram-negative bacteria. The complexes exhibit better or equal inhibition in comparison
to free fluoroquinolones. Binding interactions of the complexes with calf thymus (CT DNA) were investigated by absorption
titration, viscosity studies and DNA melting temperature experiment. The cleavage reaction on pUC19 DNA was monitored by
agarose gel electrophoresis. The lower concentration of the complexes was catalysed the dismutation of superoxide radical at
Jb oi oh lno gW i ci lae lyp &H S, ow nh si ,c Lh t di n. dicates that the complexes can act as a possible model for superoxide dismutase. Copyright ꢀc 2011
n
Supporting information may be found in the online version of this article.
Keywords: calf thymus DNA; pUC19 DNA; melting temperature; superoxide dismutase
Introduction
A large number of mixed ligand copper(II) complexes have been
shown to exhibit superoxide dismutase activity.[ This activity
depends on the Cu(II)/Cu(I) redox process, which is related to
the flexibility of the geometric transformation around the metal
centres.[ Herein, we studied the mode of coordination and
biological properties of Cu(II) complexes with second-generation
quinolone, ciprofloxacin and tridentate ligands.
8]
Transitionmetalcomplexeswiththeirvariedcoordinationenviron-
ments and versatile redox and spectral properties offer immense
scope for designing species that are suitable to bind and cleave
DNA. Metal ions can play a role in biology as counter-ions for
protein, DNA, RNA and in various biological organelles. The struc-
tural role is often manifested by maintenance of various biological
structures, whereas a functional role brings key reactivity to a
9]
[
1]
reaction centre for a protein.
Inrecentyears, therehasbeensubstantialinterestintherational
Experimental Section
design of novel transition metal complexes which bind and cleave
Materials and Instrumentation
duplex DNA with high sequential or structural selectivity.[
2–4]
An
All the chemicals and solvents were reagent grade and
used as purchased. Ciprofloxacin hydrochloride was purchased
from Bayer AG (Wuppertal, Germany). Cupric chloride di-
hydrate, pyridine-2-carboxaldehyde, pyridine-3-carboxaldehyde,
thiophene-2-carboxaldehyde, 9-anthraldehyde and CT DNA were
purchased from SD Fine Chemicals, India. Ethidium bromide (EB)
and Luria Broth were purchased from Himedia, India. Acetic acid
and EDTA were purchased from SD Fine Chemicals, India.
approach to the discovery of new metallodrugs involves binding
an organic compound of known therapeutic value to a metal-
containing fragment; this results in a metal–drug synergism in
which the metal acts as a carrier and stabilizer for the drug until it
reaches its target, while at the same time the organic drug carries
and protects the metal from side reactions in its transit towards
[
5]
other targets of biological action. Such combined effects may
result in an important enhancement of the activity of the drug and
opening of new mechanisms of action. The use of transition metal
complexes as artificial nucleases is an area of extensive research
owing to the diverse structural features and reactivity of such
complexes.[
Metal contents of the complexes were analysed gravimetrically
and volumetrically,[
10]
after decomposing the organic matter
with acid mixture (HClO4, H2SO4 and HNO3). C, H and N
elemental analyses were performed on a Perkin-Elmer 240
elemental analyser. Magnetic moments were measured by
Gouy’s method using mercury tetrathiocyanatocobaltate(II) as
6]
Quinolones, a commonly used term for the quinolonecarboxylic
acids or 4-quinolones, are a group of synthetic antibacterial agents
containing a 4-oxo-1,4-dihydroquinoline skeleton. Ciprofloxacin
−6
◦
cgs units at 20 C), on a
the calibrant (χg = 16.44 × 10
(
CPFH) [1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazine-l-
yl)quinolone-3-carboxylic acid] is a synthetic fluoroquinolone
derivative which has broad-spectrum activity against many
pathogenic Gram-negative and Gram-positive bacteria and this
antibiotic is widely used in the treatment of a variety of bacterial
∗
Correspondenceto:Mohan N. Patel,DepartmentofChemistry,SardarPatelUni-
versity,Vallabh Vidyanagar-388120, Gujarat, India. E-mail: jeenen@gmail.com
DepartmentofChemistry,SardarPatelUniversity,VallabhVidyanagar-388120,
Gujarat, India
[
7]
infections in humans and animals.
Appl. Organometal. Chem. 2011, 25, 653–660
Copyright ꢀc 2011 John Wiley & Sons, Ltd.

M. N. Patel, P. A. Dosi and B. S. Bhatt
1
Scheme 1. Structure of the title complex [Cu(cpf)(A )Cl].
•
Citizen Balance. The diamagnetic correction was made using
Pascal’s constant. IR spectra were recorded on an FT-IR Shimadzu
spectrophotometer with the sample prepared as KBr pellets in the
range 4000–400 cm . The reflectance spectra were recorded on
a Lambda 19 UV–vis–NIR spectrophotometer (Perkin Elmer, USA).
The FAB mass spectra were recorded on a Jeol SX 120/Da-600 mass
spectrometer/data system using argon/xenon (6 kV, 10 mA) as the
fast atomic bombardment (FAB) gas. The accelerating voltage was
with CH3ONa (0.27 g, 1.5 mmol), CuCl2 2H2O (0.84 g, 1.5 mmol)
ꢁ
ꢁ
ꢁ
ꢁꢁ
in CH3OH (15 mL) and 4 -(2-pyridyl)-2,2 :6 ,2 -terpyridine (1.3 g,
1.5 mmol) in CH OH (15 mL). The reaction mixture was re-
fluxed for 2 h. A fine amorphous product of green colour
was obtained, which was washed with ether/hexane and dried
in a vacuum desiccator. The proposed reaction is shown in
Scheme 1.
3
−
1
◦
Yield: 64%, m.p.: 242 C, µeff: 1.88 B.M. Anal. calcd for
1
0 kV and spectra were recorded at room temperature.
C H ClFCuN O (727.67): C, 59.42; H, 4.29; N, 13.47 Cu, 8.73.
3
6
31
7
3
Found: C, 59.39; H, 4.24; N, 13.34; Cu, 8.69%.
Synthesis of Ligands
All tridentate ligands were synthesized using a literature
procedure.[ Aqueous KOH (10 mL, 1.5 M solution) was added
to a stirred solution of 2 equiv. 2-acetylpyridine (1.20 g, 10 mmol)
and 1 equiv. aldehydes (10 mmol) in ethanol (20 ml) and ammonia
solution (10 mL). After stirring for 4 h at room temperature, the
resultant orange precipitate was isolated by filtration and recrys-
2
[
Cu(cpf)(A )Cl] [II]
11]
2
ꢁ
ꢁ
ꢁ
ꢁꢁ
[
Cu(cpf)(A )Cl] [II] was prepared using 4 -(3-pyridyl)-2,2 :6 ,2 -
◦
terpyridine (1.5 mmol). Yield: 67%, m.p.: 232 C, µeff: 1.87 B.M.
Anal. calcd for C36H31ClFCuN7O3 (727.67): C, 59.42; H, 4.29; N,
3.47 Cu, 8.73. Found: C, 59.34; H, 4.21; N, 13.41; Cu, 8.67%.
1
1
tallized from methanol. Ligands were characterized using H-NMR
1
3
and C-NMR spectra.
3
[
Cu(cpf)(A )Cl] [III]
Synthesis of Complexes
3
ꢁ
ꢁ
ꢁ
ꢁꢁ
[
Cu(cpf)(A )Cl] [III] was prepared using 4 - phenyl-2,2 :6 ,2 -
1
[
Cu(cpf)(A )Cl] [I]
◦
terpyridine (1.5 mmol). Yield: 63%, m.p.: 222 C, µeff: 1.91 B.M.
Anal. calcd for C38H32ClFCuN6O3 (738.70): C, 61.79; H, 4.37; N,
11.38; Cu, 8.60. Found: C, 61.68; H, 4.24; N, 11.30; Cu, 8.53%.
Complex 1 was prepared by the reaction of ciprofloxacin
1.8 g, 1.5 mmol) in CH3OH (10 mL), which was deprotonated
(
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Copyright ꢀc 2011 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2011, 25, 653–660

Drug-based copper(II) complexes
4
[
Cu(cpf)(A )Cl] [IV]
DNA Melting Experiment
4
ꢁ
ꢁ
ꢁ
ꢁꢁ
[
Cu(cpf)(A )Cl] [IV] was prepared using 4 -(2-thiophene)-2,2 :6 ,2 -
Thermal denaturation studies were carried out with a Perkin-
Elmer Lambda 35 spectrophotometer equipped with a Peltier
◦
terpyridine(1.5 mmol). Yield:70%, m.p.:231 C, µeff:1.84B.M. Anal.
calcd for: C36H30ClFCuN6O3S (743.11): C, 58.06; H, 4.06; N, 11.28;
Cu, 8.53. Found: C, 58.01; H, 4.02; N, 11.21; Cu, 8.45%.
◦
temperature-controlling programmer (± 0.1 C). The melting
curves were obtained by measuring the absorbance at 260 nm
for solutions of CT DNA (100 µM) in the absence and presence of
the Cu(II) complexes (20 µM) as a function of the temperature. The
5
[
Cu(cpf)(A )Cl] [V]
◦
◦
temperature was scanned from 35 to 90 C at a speed of 5 C/min.
The melting temperature (Tm) was taken as the mid-point of
hyperchromic transition.
5
ꢁ
[
Cu(cpf)(A )Cl] [V] was prepared using 4 -(anthracene-9-yl)-
ꢁ
ꢁ
ꢁꢁ
◦
2
1
4
,2 :6 ,2 -terpyridine (1.5 mmol). Yield: 70%, m.p.: 234 C, µeff:
.85 B.M. Anal. calcd for C46H36ClFCuN6O3 (838.81): C, 65.87; H,
.33; N, 10.02; Cu, 7.58. Found: C, 65.75; H, 4.31; N, 10.05; Cu, 7.50%.
Gel Electrophoresis
The plasmid DNA used was extracted from E. coli specie belonging
In-vitro Antibacterial Activity
to pUC19 genus as per the standard protocol reported by
An antibacterial activity assay was performed on Staphylococcus
aureus, Bacillus subtilis, Serratia marcescens, Pseudomonas aerug-
inosa and Escherichia coli. The antibacterial activity for the test
compounds was tested to determine the bacteriostatic concen-
tration, i.e. minimum inhibitory concentration (MIC) in terms
micromoles. The MIC value was determined by broth dilution
[15]
Sambrook and Russell.
following three steps:
The whole process was based on the
(1) weakening of the bacterial cell wall by the action of lysozyme.
(2) cells being lysed by ethylenediaminetetraaccetate EDTA and
detergent at high pH;
technique.[
12]
(3) insoluble cells debris consisting of genomic DNA and
protein being precipitated in high salt-containing buffer,
leaving behind the plasmid in solution (supernant), which is
precipitated by chilled absolute alcohol and collected.
A preculture of bacteria was grown in LB (Luria
Broth) overnight at the most favourable temperature of each
species. This culture was used as a control to examine if the
growth of bacteria tested was normal. In a similar second culture,
2
0 µL of the bacteria as well as the tested compound at the desired
Agarose gel electrophoresis experiments were carried out on
supercoiled plasmid DNA pUC19 using a horizontal gel system.
The DNA cleavage profile was analysed using 1% agarose gel
in a horizontal gel tank set with a running time of 2.5 h, at a
constant voltage of 50 V in 1× Tris-acetate EDTA (TAE) buffer
concentration were added and monitored for bacterial growth by
measuring turbidity of the culture after 18 h. If a certain con-
centration of a compound inhibited bacterial growth, half of the
concentration of the compound was tested. This procedure was
carried out up to the concentration which inhibited the growth of
bacteria. The lowest concentration that inhibited bacterial growth
was considered the MIC value. All equipments and culture media
were sterilized.
(
0.04 M Tris–acetate, pH 8, 0.001 M EDTA) by loading each reaction
mixture(15 µL)consisting ofplasmidDNA(150 µg/mL)inTEbuffer
(
10 mM Tris, 1 mM EDTA, pH 8.0) and 200 µM complex, which were
◦
allowed to proceed for 24 h at 37 C and quenched by addition
of 5 µL loading buffer (40% sucrose, 0.25% bromophenol blue,
0.25% xylene cyanole FF, 200 mM EDTA). The gel was stained with
Viscosity Measurement
0
.5 µg/mL of ethidium bromide and was photographed on a UV
Viscometric titrations were performed with an Ubbelohde auto-
mated viscometer. The viscometer was thermostated at 37 C in a
trans-illuminator. The extent of cleavage was quantified based on
the intensity of the band using AlphaDigiDoc RT. The degree of
DNA cleavage activity was expressed in terms of the percentage
of conversion of the SC-DNA to open circular (OC)-DNA according
to the following equation:[
◦

constant temperature bath. The concentration of DNA was 200 µM
in nucleotide phosphate (NP) and the flow times were measured
with an automated timer. Each sample was measured three times,
and an average flow time was calculated. Data were presented
16]
1
/3
as (η/η0)
versus [complex]/[DNA], where η is the viscosity of
%
DNA cleavage activity
DNA in presence of complex and η0 that of DNA alone. Viscosity
values were calculated from the observed flowing time of DNA-
containing solutions (t) corrected for that of buffer alone (t0),
ꢀ
(% of SC-DNA)control − (% of SC-DNA)sample]ꢁ
[
=
× 100
(% of SC-DNA)control
[
13]
η = (t − t0).
Absorption Titration
Superoxide Dismutase Activity
The UV absorbance at 260 and 280 nm of the CT DNA solution
in 5 mM Tris–HCl buffer (pH 7.2) gave a ratio of 1.9, indicating
that the DNA was free of protein. The concentration of CT DNA
was measured from the band intensity at 260 nm with a known
The superoxide dismutase (SOD) activities of the copper com-
pounds were determined spectrophotometrically by the nitroblue
tetrazolium (NBT) assay method.[ One unit of SOD activity was
defined as the concentration of the test substrate required for 50%
inhibition of NBT reduction by superoxide anion (IC50 value).
The superoxide radial anion produced by 79 µM (nicotinamide
adenine dinucleotide reduced) NADH, 30 µM phenazine metho-
sulphate (PMS) and 75 µM NBT in phosphate buffer (pH = 7.8). The
concentration of tested compounds varied from 0.25 to 3.0 µM.
The amount of reduced NBT was spectrophotometrically detected
by monitoring the concentration of the blue formazan form, which
17]
−
1
−1 [14]
value (6600 M cm ). Absorption titration measurements
were carried out by varying the concentration of CT DNA, keeping
the metal complex concentration constant in 5 mM Tris–HCl/5 mM
NaCl buffer (pH 7.2). Samples were kept for equilibrium before
recording spectrum. The intrinsic binding constants (Kb) were
determined from a plot of [DNA]/(εa − εf) versus [DNA] using
absorption spectral titration data.
Appl. Organometal. Chem. 2011, 25, 653–660
Copyright ꢀc 2011 John Wiley & Sons, Ltd.
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M. N. Patel, P. A. Dosi and B. S. Bhatt
to typical high-spin octahedral complexes. However, the values
are slightly higher than the expected spin-only values owing to
the spin–orbit coupling contribution.[
Table 1. IR spectra data (cm 1
−
)
19]
ν(C O)
Compounds pyridone ν(COO)as ν(COO)s ꢀν ν(M–N) ν(M–O)
Ciprofloxacin
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
1708
1616
1615
1622
1624
1618
1624
1579
1577
1584
1585
1581
1340 284
1374 205
1381 196
1382 198
1382 198
1375 206
–
IR Spectra
535
531
543
538
545
512
505
510
517
511
The IR spectra of ligands are quite complex owing to the
presence of numerous functional groups in the molecules.
The characterization of metal complexes can be achieved by
studying the most typical vibrations that are characteristic of
the coordination sites of ligands. The determination of the
coordinating atoms was made on the basis of the comparison
of the IR spectra of the ligands and complexes Significant wave
numbers are given in Table 1.
absorbs at 560 nm. The reduction rate of NBT was measured in
the presence and absence of test compounds at various concen-
trations of complex in the system. All measurements were carried
out at room temperature. The percentage inhibition (η) of NBT
reduction was calculated using the following equation:
1) Theν(C O)stretchingvibrationbandappearedat1708 cm 1
−
(
for ciprofloxacin, whereas for complexes it appeared at
−
1
1
615–1624 cm . This shift towards lower energy suggests
thatcoordinationoccursthroughcarbonyloxygenofpyridine
ring.
2) Strong absorption band at 1624 and 1340 cm 1 in
ciprofloxacin could be assigned to ν(COO) asymmetric
and symmetric vibration, respectively, while in metal com-
plexes these bands were observed at 1577–1585 and
ꢁ
η(% inhibition of NBT reduction) = (1 − k /k) × 100
−
(
ꢁ
where k and k represent the slopes of the straight line of
absorbance values as a function of time in the presence and
absence of SOD mimic or a model compound, respectively.
Concentration of the complex which causes 50% inhibition in the
reductionrateofNBTisreportedastheIC50 valueofthecomplexes,
determined by plotting the graph of percentage inhibition of NBT
reduction against increase in concentration of the complex.
−
1
1
374–1382 cm . The difference ꢀν = νas(COO)–νs(COO)
is useful for determining the coordination mode of ligands.
The ꢀν values were greater than 200 cm , indicating the
−
1
[
20–24]
monodentatecoordinationmodeofcarboxylatogroup.
These data are further supported by ν(M–O) which appears
−
1
at 505–517 cm for complexes.
(
3) In investigated complexes the ν(C N) band of terpyridines
Results and Discussion
−
1
appeared at ∼1584 cm . This band shifted to a higher
frequency at ∼1626 cm in complexes, indicating tri-
−
1[25,26]
Reflectance Spectra and Magnetism
dentate N–N coordination of the ligand. The N → M bonding
The diffuse reflectance spectra of Cu(II)–ciprofloxacin complexes
consisted of one asymmetric broad band around 15 000 cm
These spectra suggest an octahedral geometry of the ligands
donor atoms around the cupric ion.
[27]
−1
was supported by the ν(M–N) band
for complexes.
at ∼531–545 cm
−
1
.
[
18]
The magnetic moments of the copper(II) complexes lie in the
Mass Spectra
1
.84–1.91 B.M. range. These values are typical of mononuclear
9
copper(II) compounds with d electronic configuration. The
observed magnetic moments of all the complexes correspond
Figure 1 represents the FAB – mass spectrum of complex I, that is
1
[Cu(cpf)(A )Cl], obtained using m-nitro benzyl alcohol as matrix.
1
Figure 1. FAB-mass spectrum of complex 1, that is [Cu(cpf)(A )Cl], obtained using m-nitro benzyl alcohol.
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Copyright ꢀc 2011 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2011, 25, 653–660

Drug-based copper(II) complexes
Table 2. Minimum inhibitory concentration data of the compounds (µM)
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
CuCl2·2H2O
2698.00
1.6
2815.00
1.1
2756.00
1.6
2404.00
1.4
3402.00
1.4
Ciprofloxacin
Gatifloxacin
5.1
4.0
2.9
1.0
2.9
Norfloxacin
2.5
2.5
4.1
3.8
2.8
Enrofloxacin
1.9
3.9
1.7
1.4
1.4
Pefloxacin
2.1
2.4
5.1
5.7
2.7
Levofloxacin
1.7
2.2
1.7
1.7
1.0
Sparfloxacin
1.3
2.0
1.5
1.5
1.3
Ofloxacin
1.9
1.4
1.7
2.2
1.4
Ligand 1 (A1)
550
575
700
550
650
2.8
500
575
750
575
650
2.3
550
575
750
650
750
2.7
525
550
725
650
725
2.7
550
550
750
650
750
2.7
Ligand 2(A2)
Ligand 3 (A3)
Ligand 4 (A4)
Ligand 5 (A5)
Ligand 1 + ciprofloxacin
Ligand 2 + ciprofloxacin
Ligand 3 + ciprofloxacin
Ligand 4 + ciprofloxacin
2.9
2.4
2.9
2.7
2.8
3.5
2.9
3.3
3.2
3.3
2.9
2.4
3.2
3.1
3.1
Ligand 5 + ciprofloxacin
3.3
2.9
3.3
3.0
3.3
1
[
[
[
[
[
Cu(cpf)(A )Cl] (1)
1.1
0.9
0.4
0.4
1.2
2
Cu(cpf)(A )Cl] (2)
0.7
0.9
1.8
0.9
1.5
3
Cu(cpf)(A )Cl] (3)
1.2
1.1
1.7
1.4
1.1
4
Cu(cpf)(A )Cl] (4)
0.8
1.0
0.6
1.1
1.0
5
Cu(cpf)(A )Cl] (5)
1.2
0.7
1.7
1.2
1.0
Peaks at 136, 137, 154, 289 and 307 m/z value were due to the
usage of matrix. Peaks at 726 and 728 in spectra were assigned to
activity of all the complexes is even higher than the antibacterial
activity of complexes, which were previously reported by our
+
[28]
(
M) and (M + 2) of the complex molecule associated with four H
group.
It has also been suggested[29,30] that the ligands with nitrogen
donor systems might inhibit enzyme production, since the
enzymes that require these groups for their activity appear to
ions in the absence of lattice water. There also existed a doublet at
m/z = 564 for a fragment of the complex (m/z = 428) rid of the
neutralligandassociatedwiththematrix(m/z = 136).Thedoublet
nature observed in case of the fragment suggests the presence of
one Cl atom. Loss of the chlorine atom gave a fragment ion peak at
m/z = 691, which also confirms that the chlorine atom attached
to the metal ion with a covalent bond. Several other fragments at
be especially susceptible to deactivation by the metal ions upon
chelation. Chelation reduces the polarity[
29,30]
of the metal ion,
mainly because of the partial sharing of its positive charge with the
donorgroupsandpossiblytheπ-electrondelocalizationwithinthe
whole chelate ring system thus formed during coordination. This
process of chelation increases the lipophilic nature of the central
metalatom,whichinturnfavoursitspermeationthroughthelipoid
layer of the membrane, increasing the hydrophobic character and
liposolubility of the molecule in crossing the cell membrane of
the microorganism, and hence enhances the biological utilization
ratio and activity of the testing drugs/compounds.
6
01, 396, 361, 331 and 323 m/z value were observed. The isotopic
pattern owing to the presence of chlorine and copper appeared
in the spectrum.
In-vitro Antibacterial Activity
The efficiency of the ligands and the complexes was tested against
three Gram-negative (E. coli, S. marcescens and P. aeruginosa) and
two Gram-positive (S. aureus and B. subtilis) microorganisms. The
results of the minimum inhibitory concentration (MIC), expressed
in micromoles, are presented in Table 2.
Electronic Absorption Titration
In case of S. aureus, all compounds showed good activity
comparedwithreferencedrugs. IncaseofB.subtilis,allcompounds
exhibited good activity compare to reference drugs. In case of S.
marcescens, compounds I and IV were found to be more potent
than the reference drugs. In the case of P. aeruginosa, compounds
I, II, IV and V exhibited good activity compared with the reference
drugs. In the case of E. coli, compounds I and III–V showed more
potency than the reference drugs. The complexes showed better
antimicrobial activity than the free ligands and ciprofloxacin.
The higher antimicrobial activity can be mainly attributed to the
existence of the quinolone in the complexes. The antibacterial
DNA can provide three distinctive binding sites for quinolone-
metal complexes, namely groove, phosphate group and
intercalation.[
31,32]
This behavior is of great importance with re-
gard to the relevant biological role of quinolone antibiotics in the
body.[
33–35]
Electronic absorption spectra are universally employed to
determine the binding of complexes with DNA. Complex bound
to DNA through intercalation usually results in hypochromism
and red shift (bathchromism). The extent of the hypochromism
is commonly consistent with the strength of intercalative
interaction.[
36]
Appl. Organometal. Chem. 2011, 25, 653–660
Copyright ꢀc 2011 John Wiley & Sons, Ltd.
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M. N. Patel, P. A. Dosi and B. S. Bhatt
1
Figure 2. Electronic absorption titration curve of [Cu(cpf)(A )Cl] in absence
and in presence of increasing amount of DNA; 50–150 µM in phosphate
buffer in 5 mM Tris-HCl buffer (pH 7.2), [complex] = 15 µM, with incubation
◦
period of 30 min. at 37 C, Inset: Plot of [DNA]/(εa − εf ) versus [DNA].
The concentration of CT DNA was determined from the
−
1
−1
absorption intensity at 260 nm with an ε value of 6600 M cm .
From Fig. 2 absorption titration experiments were made using
different concentrations of CT DNA, while keeping the complex
concentration constant. Due correction was made for the
absorbance of the CT DNA itself. Samples were equilibrated before
recording each spectrum. For complexes, the intrinsic binding
constant, Kb was been determined from spectral titration data
Figure 3. Effect on relative viscosity of DNA under the influence of
◦
increasing amount of complexes at 27 ± 0.1 C in 5 mM Tris-HCl buffer (pH
7.2) as a medium.
[
37]
using following equation:
[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb(εb − εf)
where εa, εf, and εb correspond to Aobsd/[Cu(II) complex], the
extinction coefficient for the free complex, and the extinction
coefficient for complex in fully bound form, respectively.
The binding constants obtained for complexes I–V were
4
4
4
4
4
−1
,
3
.33 × 10 , 3.12 × 10 , 2.33 × 10 , 2.71 × 10 and 2.54 × 10
M
respectively. The Kb values of complexes were similar to that of
2
+
4
−1
[38]
[
Ru(bpy)2ppd] (2.18 × 10
M
) complex.
The Kb value of
I and II is higher than that for most of the previously reported
n
[28]
complexesoftype[Cu(cpf)(A )Cl]. Thesespectralcharacteristics
are consistent with a mode of interaction that involves a stacking
interaction between the complex and the base pairs of DNA, which
means that the complexes can intercalate into the double helix
structure of DNA.
Figure 4. Melting curves of CT DNA in the absence and presence of
complexes 1–5.
Viscosity Measurement
DNA Melting Experiment
The binding mode of complexes to DNA was explained by
viscosity measurement. Hydrodynamic measurements, which are
sensitive to the increase in length of DNA, are regarded as
the least ambiguous and the most critical tests in absence of
The melting temperature (Tm) of double-stranded DNA changes
with different binding modes. Generally, the melting temperature
increases when metal complexes bind to DNA by intercalation, as
intercalation of the complexes into DNA base pairs causes stabi-
lization of base stacking and hence raises the melting temperature
of the double-stranded DNA. DNA melting experiments are useful
in establishing the extent of intercalation.[ A large change in the
Tm of DNA is indicative of a strong interaction with DNA. The effect
of complexes on the melting temperature of CT DNA in buffer
is shown in Fig. 4. At the melting temperature, the double helix
denatures into single-stranded DNA. The thermal denaturation
experiment carried out for DNA in the absence of complex gave
[
39,40]
crystallographic structure data.
A classical intercalation leads
to lengthening of DNA helix because of the separation of base
pairs to accommodate the ligand, and thus the viscosity of DNA
increases while a partial nonclassical intercalation could bend
the DNA helix and thus reduce its effective length along with its
viscosity.[
42]
41]
The viscosity of DNA is increased with increasing amounts
of complexes I–V (Fig. 3). Upon increasing the amounts of
complexes, the relative viscosity of DNA increases steadily, less
than the classical intercalator EB. The increased degree of viscosity,
which may depend on the affinity for DNA, follows the order
EB > I > II > IV > V > III > ciprofloxacin. The viscosity results
show that all complexes intercalate through classical intercalation
mode.
◦
a Tm74.2 ± 1 C under our experimental conditions. The observed
◦
melting temperaturesinthepresenceofcomplexwere79.0± 1 C,
◦
◦
78.5 ± 1 C and 78.4 ± 1 C for complexes I, II and IV, respectively,
and for complexes III and V the Tm increased to 77.0 ± 1 and
◦
◦
78.2 ± 1 C respectively. The ꢀTm (3.8–4.6 C) values of the DNA
increased in the presence of the complexes and were comparable
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Copyright ꢀc 2011 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2011, 25, 653–660

Drug-based copper(II) complexes
Figure 5. Photogenic view of interaction of pUC19 DNA (450 µg/mL) with series of copper(II) complexes (200 µM) using 1% agarose gel containing
◦
0
.5 µg/mL ethidium bromide. All reactions were incubated in TE buffer (pH 8) in a final volume of 15 µL, for 3 h. at 37 C.: Lane 1, DNA control; Lane 2,
•
1
2
4
5
CuCl2 2H2O; Lane 3, ciprofloxacin; Lane 4, [Cu(cpf)(A )Cl]; Lane 5, [Cu(cpf)(A )Cl]; Lane 6, [Cu(L)(cpf)Cl]; Lane 7, [Cu(cpf)(A )Cl]; Lane 8[Cu(cpf)(A )Cl].
Table 3. Gel electrophoresis data
%
DNA
Compounds
% SC
% OC
% L
cleavage
DNA control
DNA + metal salt
DNA + ciprofloxacin
DNA + I
76
71
65
21
23
28
24
27
24
29
35
47
43
45
43
45
–
–
–
6.57
–
14.47
72.36
69.73
63.15
68.4
32
34
27
34
28
DNA + II
DNA + III
DNA + IV
DNA + V
64.47
to those observed for classical intercalator,[43] suggesting the large
DNA-binding affinity of the complexes.
Figure 6. plot of absorbance values (Abs560) against time (t).
Gel Electrophoresis
DNA cleavage accelerated by transition metal complexes is the
centre of interest.[
44,45]
The cleavage of plasmid pUC19 DNA was
monitored by gel electrophoresis to investigate the ability of the
copper(II) complexes to serve as metallonucleases. The naturally
occurring supercoiled form (form I), when nicked, gives rise to an
open circular relaxed form (form II) and further cleaves to a linear
form (form III). From Fig. 5, when subjected to gel electrophoresis,
form I shows the fastest migration compared with forms II and
III. Form II migrates very slowly prior to its relaxed structure,
whereas form III migrates somewhere between the positions of
form I and form II. These clearly show that the relative binding
efficacy of complexes to DNA is much higher than the binding
efficacy of metal salt or ciprofloxacin (Table 3). The difference in
DNA–cleavage efficiency of complexes was due to the difference
in binding affinity of complexes to DNA.
Figure 7. Plot of percentage of inhibiting NBT reduction with an increase
in the concentration of complex 1.
Table 4. IC50 values of copper(II) complexes
Complexes
IC50 (µM)
Superoxide Dismutase Activity
1
[
[
[
[
[
Cu(cpf)(A )Cl] (1)
0.5
The SOD activity of the complexes was investigated by NBT assay.
Several complexes containing transition metals,
copper, are known to give good SOD activity, although their
structures are totally unrelated to the native enzyme.
the SOD activity was measured at pH 7.8. The concentration
required to yield 50% inhibition of the reduction rate of NBT
2
Cu(cpf)(A )Cl] (2)
0.75
1.25
1.0
[
46]
including
3
Cu(cpf)(A )Cl] (3)
4
Cu(cpf)(A )Cl] (4)
[
47]
Herein,
5
Cu(cpf)(A )Cl] (5)
1.05
(IC50) was higher. The observed higher values may be due to the
presence of α-triimine ligands (terpyridine) and greater ligand
field crowding over the central metal ion.
The percentage inhibition of formazan formation at various
concentrations of complexes as a function of time was measured
by measuring the absorbance at 560 nm and plotted to a straight
line (Fig. 6). As the concentration of tested complexes increased,
the slope (m) decreased. Percentage inhibition of the reduction
of NBT was plotted against the concentration of the complex
(Fig. 7). Compounds exhibited SOD-like activity at biological pH
with the IC50 values in the range 0.5–1.25 µM, which is very
similar for the complexes of same type reported previously by our
group.[ The superoxide scavenging data are shown in Table 4.
28]
Appl. Organometal. Chem. 2011, 25, 653–660
Copyright ꢀc 2011 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

M. N. Patel, P. A. Dosi and B. S. Bhatt
The results show that complexes exhibit greater scavenging
activity towards superoxide radicals, which may be accredited
to the redox potential of Cu(II) complex, which depends on the
geometry at the metal centre. The mechanism for scavenging of
ROS may move forward via an unstable octahedral adduct under
[15] J. Sambrook, D. W. Russell, Preparation of Plasmid DNA by Alkaline
Lysis with SDS: Minipreparation, Molecular Cloning, A Laboratory
Manual, 3rd edn, Vol. 1, Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, NY.
[16] J. Yang, R. N. S. Wong, M. S. Yang, Chem.-Biol. Interact. 2000, 125,
221.
[
48]
influence of the Jahn–Teller effect. Hence, there is a possibility
of rapid interconversion of Cu(II) and Cu(I) via electron transfer
between copper and reactive oxygen radical anions following the
[17] V. Ponti, M. V. Dianzaini, K. J. Cheesoman, T. F. Stater, Chem.-Biol.
Interact. 1978, 23, 281.
[
18] B. N. Figgis, J. Lewis, in Modern Coordination Chemistry: Principles
and Methods (Eds.: J. Lewis, R. G. Wilkins), Interscience: New York,
[
49,50]
principle of electroneutrality.
1
960, p. 400.
[
[
19] F. A. Cotton, G. Wilkinson Advanced Inorganic Chemistry, 5th edn,
Wiley: New York, 1988.
20] K. Nakamoto, Infrared and Raman Spectra of Inorganic and
Coordination Compounds, 4th edn, Wiley Interscience: New York,
Conclusion
1
986.
The binding behaviours of the copper complexes of terpyridines
and ciprofloxacin were found to be subtly but distinctly different,
depending on the structure of terpyridines. The increasing order
for the interacting behaviour of synthesized complexes was
III < V < IV < II < I on the bases of Kb values, change in relative
viscosity and plasmid cleavage study. The ligand can facilitate the
stabilization of bonding between metal centre and the oxygen
radicalanion, favouringtheenhancementofenzymaticbehaviour.
[
[
21] J. R. Anacona, I. Rodriguez, J. Coord. Chem. 2004, 57, 1263.
22] G. B. Deacon, R. Philips, J. Coord. Chem. Rev. 1980, 33, 227.
[23] Z. H. Chohan, C. T. Suparan, A. Scozzafava, J. Enz. Inhib. Med. Chem.
005, 23, 303.
2
[
24] N. H. Patel, P. K. Panchal, P. B. Pansuriya, M. N. Patel, J. Macro. Sci.:
Part A Pure Appl. Chem. 2006, 43, 1083.
25] P. B. Pansuriya, P. Dhnadhukia, V. Thakkar, M. N. Patel, J. Enz. Inhib.
Med. Chem. 2007, 22, 477.
[
[26] P. K. Panchal, M. N. Patel, Synth. React. Inorg. Met.-Chem. 2004, 34,
277.
27] H. H. Freedman, J. Am. Chem. Soc. 1961, 83, 2900.
[28] M. N. Patel, P. A. Dosi, B. S. Bhatt, Polyhedron 2010, 29, 3238–3245.
29] Z. H. Chohan, App. Organomet. Chem. 2006, 20, 112.
1
[
Acknowledgements
[
The authors thank the Head of the Department of Chemistry,
Sardar Patel University, India for making it convenient to work
in the laboratory and UGC for providing financial support under
[30] Z. H. Chohan, M. Arif, Z. Shafiq, M. Yaqub, C. T. Supuran, J. Enz. Inhib.
Med. Chem. 2006, 21, 95.
[31] A. Rodger, B. Norden (Eds.), Circular Dichroism and Linear Dichroism,
Oxford University Press: Oxford, 1997.
‘‘UGC Research Fellowship for Meritorious Students’’ scheme.
[
[
[
[
32] B. M. Zeglis, V. C. Pierre, J. K. Barton, Chem. Commun. 2007, 4565.
33] G. Song, Y. He, Z. Cai, J. Fluoresc. 2004, 14, 705.
34] P. Drevensek, I. Turel, N. P. Ulrih, J. Inorg. Biochem. 2003, 96, 407.
35] G. S. Son, J.-A. Yeo, M.-S. Kim, S. K. Kim, A. Holmen, B. Akerman,
B. Norden, J. Am. Chem. Soc. 1998, 120, 6451.
Supporting information
Supporting information may be found in the online version of this
article.
[
36] T. M. Kelly, A. B. Tossi, D. J. McConnell, T. C. Strekas, Nucl. Acids Res.
1
985, 13, 6017.
[
[
37] M. T. Carter, M. Rodriguez, A. Bard, J.Am.Chem.Soc. 1989, 111, 8901.
38] B. Peng, H. Chao, B. Sun, H. Li, F. Gao, L. N. Ji, J. Inorg. Biochem. 2006,
100, 1487.
References
[
39] J. K. Barton,A. T. Danishefsky,J. M. Goldberg,J.Am.Chem.Soc.1984,
106, 2172.
[
[
[
[
[
1] D. C. Crans, J. J. Smee, E. Gaidamauskas, L. Yang, Chem. Rev. 2004,
04, 849.
2] B. H. Geierstanger, M. Marksich, P. B. Dervan, D. E. Wemmer, Science
994, 266, 646.
3] C. Liu, J. Zhou, Q. Li, L. Wang, Z. Liao, H. Xu, J. Inorg. Biochem. 1999,
5, 233.
4] G. Prativiel, J. Bernadou, B. Meunier, Angew. Chem., Int. Ed. Engl.
995, 34, 746.
5] R. A. S a´ nchez-Delgado, A. Anzelloti, Mini Rev. Med. Chem. 2004, 4,
3.
1
[40] A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar, N. J. Turro,
J. K. Barton, J. Am. Chem. Soc. 1989, 111, 3051.
[41] H. Deng, J. Li, K. C. Zheng, Y. Yang, H. Chao, L. N. Ji, Inorg. Chim. Acta
2005, 358, 3430.
[42] J. M. Kelly, A. B. Tossi, D. J. McConnell, C. Ohuigin, Nucl. Acids Res.
1985, 13, 6017.
[43] M. J. Han, L. H. Gao, Y. X. Lu K. Z. Wang, J. Phys. Chem. B, 2006, 110,
2364.
[44] R. P. Hertzberg, P. B. Dervan, J. Am. Chem. Soc. 1982, 104, 313.
[45] D. S. Sigman, D. R. Graham, L. E. Marshall, K. A. Reich, J. Am. Chem.
Soc. 1980, 102, 5419.
[46] R. Rajan, R. Rajaram, B. U. Nair, T. Ramasami, S. K. Mandal, J. Chem.
Soc., Dalton Trans. 1996, 2019.
[47] U. Weser, L. M. Schubotz, E. Lengfelder, J. Mol. Catal. 1981, 13, 249.
[48] F. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, Wiley: New
York, 1972.
1
7
1
2
[
[
[
[
6] D. S. Sigman, A. Mazumder, M. D. Perrin, Chem. Rev. 1993, 93, 2295.
7] A. I. Darkopoulos, P. C. Ioannou, Anal. Chem. 1997, 354, 197.
8] A. L. Abuhijleh, J. Inorg. Biochem. 1997, 68, 167.
9] J. C. Casanova, G. Alzuci, J. Borras, J. Latorre, M. Sanau, S. G. Granda,
J. Inorg. Biochem. 1995, 60, 219.
[10] A. I. Vogel, TextbookofQuantitativeInorganicAnalysis, 4th edn, ELBS
and Longman: London, 1978.
[
[
11] G. S. Hanan, J. Wang, Synlett. 2005, 1251.
12] D. Sinha, A. K. Tiwari, S. Singh, G. Shukla, P. Mishra, H. Chandra,
A. K. Mishra, Eur. J. Med. Chem. 2008, 43, 160.
13] G. Cohen, H. Eisenberg, Biopolymers 1969, 8, 45.
14] R. Rao, A. K. Patra, P. R. Chetana, Polyhedron 2008, 27, 1343.
[49] J. A. Fee, MetalIons in Biological Systems (Ed.: H. Siegel), Vol. 13,
Marcel Dekker: New York, 1981.
[50] J. E. Huheey,InorganicChemistryPrinciplesofStructureandReactivity,
3rd edn, Harper international: London, 1983.
[
[
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Copyright ꢀc 2011 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2011, 25, 653–660